In spite of numerous advances in medical research, cancer remains as one of the leading causes of death in the United States. In industrialized nations, roughly one in five persons will die of cancer. Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The only hope for long-term survival for HCC patients is surgical resection or liver transplantation. See Wong et al., “Circulating Tumor Cell mRNAs in Peripheral Blood From Hepatocellular Carcinoma Patients Under Radiotherapy, Surgical Resection or Chemotherapy: A Quantitative Evaluation,” Cancer Letters, 167: 183–191 (2001). However, only a minority of HCC patients are cured by removal of the tumor either by resection or transplantation and for the majority of patients, the current treatments remain unsatisfactory and the prognosis is poor. Further, more than half of those who apparently undergo successful resection subsequently develop recurrence. See Wong et al., supra, and Wong et al., “Hematogenous Dissemination of Hepatocytes and Tumor Cells After Surgical Resection of Hepatocellular Carcinoma: A Quantitative Analysis,” Clinical Cancer Research, 5: 4021–4027 (1999).
Alpha-fetoprotein is a glycoprotein that is normally expressed during embryogenesis. It is primarily expressed in liver cells, as well as in the gut, stomach, trophoblast, lungs and pancreas. The concentration of AFP in serum decreases as the liver develops and matures. AFP levels, though, can become elevated in some disease states, particularly in HCC. See Jiang et al., “Detection of Alphafetoprotein-expressing Cells in the Blood of Patients with Hepatoma and Hepatitis,” British Journal of Cancer, 75(6): 928–933 (1997). As such, elevated serum AFP levels have been employed as a highly specific and sensitive marker for the diagnosis of HCC. See Jiang et al., supra. Further, it has recently been found that substantially elevated levels of AFP mRNA in circulating blood are associated with recurrence or metastasis of HCC. See Wong et al., “Quantitative Comparison of Alpha-fetoprotein and Albumin mRNA Levels in Hepatocellular Carcinoma/Adenoma, Non-tumor Liver and Blood: Implications in Cancer Detection and Monitoring,” Cancer Letters, 156: 141–149 (2000).
Survival after the onset of symptoms of HCC is only a few months and therefore, it is important to establish techniques for the early diagnosis of HCC. As HCC patients have been shown to have elevated levels of AFP mRNA, an exemplary assay for detecting the presence and level of AFP mRNA is certainly important in the diagnosis and treatment of HCC.
Currently, there are various assays on the market to detect alpha-fetoprotein such as enzyme-linked immunosorbant assay (ELISA). However, there are problems with assays such as ELISA. For instance, with ELISA, the protein level does not always correlate with the clinical stage of HCC. Molecular biological techniques such as RT-PCR assays have also been developed. However, with such biological techniques, thermal cyclers are necessary and DNA contamination is a concern.
The present invention provides for the use of isothermal nucleic acid sequence based amplification (NASBA) which is far more advantageous than an RT-PCR assay. In particular, NASBA is an isothermal amplification process and therefore, thermal cyclers are not necessary. Further, NASBA is specific for RNA and thus, DNA contamination is not a concern.
In addition, an isothermal transcription based amplification method, as compared to RT-PCR or other amplification methods, requires few manipulations by the experimenter since it is essentially isothermal. The method may be used on purified or semi-purified RNA extracts, or on cell or tissue samples with in situ amplification. Additionally, if the sample contains both DNA and RNA, the use of RT-PCR requires a first step of DNase treatment, or some method to distinguish the amplification products of mRNA- and DNA-derived PCR products is necessary. DNase treatment prior to RT-PCR can be employed (Bitsch et al., J. Infect. Dis. 167: 740–743 (1993); Meyer et al., Mol. Cell Probes, 8: 261–271 (1994)), but it sometimes fails to remove contaminating DNA sufficiently (Bitsch et al., supra).
An isothermal amplification method for the detection or quantitation of AFP mRNA has not been described. Thus, there is a need in the art for an isothermal amplification method to detect the presence of AFP mRNA and hence detect HCC, metastasis thereof and cancer recurrence.